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Western Blot
Ben Carter, revised 7/11/2016 Materials Required ExpressPlus PAGE Gels'' (GenScript Cat# M42010) or equivalent Hoefer Mighty Small II (Fisher Cat# 03-500-483) or equivalent Immobilon-FL membrane (Fisher Cat# IPFL00010) Whatman 3mm Chromatography Paper (Fisher Cat# 05-714-5) Pharmacia Biotech TE22 transfer apparatus or equivalent Western blotting boxes (LI-COR Cat# 929-97205) or equivalent LI-COR Odyssey imager or equivalent Desired antibodies for detection '''Electrophoresis' 1. Remove the protective strip from the PAGE gel and place it in the electrophoresis apparatus. Fill the reservoir behind the gel plate with MOPS running buffer (provided as a powder with the gels). Fill the bottom basin of the apparatus to approximately 1/3rd full. 2. Load the desired amount of protein samples into the wells. Suggested: 50-200µg of protein from Arabidopsis or zebrafish tissues. 3. Perform electrophoresis at 140V for 55min. If you are using a different gel system, the voltage and duration will likely need to be adjusted. Transfer to Membrane 4. Cut the Immobilon-FL membrane slightly larger than the area of the gel. Wet the membrane in methanol for 15s. 5. Soak the membrane for 2min in Milli-Q water. Pour a square glass casserole dish or comparable basin about ½ full with freshly prepared Transfer Buffer. Remove the gel from the casting apparatus and cut the lower left corner. Soak the gel and membrane for 30min in Transfer Buffer. 6. Cut two pieces of Whatman chromatography paper to a size that can easily accommodate the membrane. Thoroughly wet one piece of Whatman paper in the Transfer Buffer. Place the membrane in the middle of the wet Whatman paper. 7. Place the gel on the top of the membrane, being careful to avoid making bubbles between the gel and the membrane. Cut the corner of the membrane that corresponds to the cut corner of the gel. 8. Wet the other piece of Whatman paper and place it on top of the stack. Using a flat object such as a gel spacer, apply gentle pressure to the smooth out any bubbles that may remain. 9. Wet two transfer sponges in the transfer buffer and assemble the transfer apparatus as follows: Transfer "Sandwich" * Cassette black side (top) * Wet sponge * Whatman paper 3mm * Gel * Membrane * Whatman paper 3mm * Wet sponge * Cassette gray side (bottom) 10. Place a rubber band around the cassette to keep it together. Place the cassette in the transfer apparatus with the cassette hinge facing the top. Fill the apparatus to the “max fill” line with Transfer Buffer. 11. Place the apparatus on a stir plate with a stir bar and apply gentle stirring. Place the lid on the apparatus with the electrodes oriented such that the gray side of the cassette is facing the red positive electrode. Run the transfer at 80V for 60min. 12. Clean a black LI-COR blotting box using the following rinses: methanol, distilled water, isopropanol. Dry the box with a lint-free wipe until no dampness remains. 13. Fill the cleaned black LI-COR box with 10-20mL of 1X TBS. Place the membrane in the box, making sure that it is entirely submerged in the TBS. You may need to trim the membrane using scissors to make it fit, but make sure to maintain the clipped corner. Rock the membrane in the TBS for 5min to remove residual polyacrylamide. 14. Place the membrane on a lint-free wipe or in the lid of the LI-COR box in a laminar flow hood and allow it to dry for 1h. Alternatively, can dry the membrane overnight in a drawer or on the benchtop. Note: If the membrane needs to be cut for subsequent steps, this is a convenient time to do so. Western Blot Note: Use 800CW secondary antibodies primarily. 680CW antibodies should be used to detect the more plentiful protein in two-color blots only as it has much higher background. For two-color blots, both antibodies can be used simultaneously in the primary antibody step and in the secondary antibody step. 15. Remove the dried membrane from the LI-COR box using gloves or forceps and wet it in a basin of methanol for 1min. Rinse with Milli-Q water. Fill the LI-COR box with 10-20mL of 1X TBS and let the membrane soak in it for 2min. 16. Pour out the TBS and replace it with 10mL of Odyssey Blocking Solution (TBS). Rock the membrane for 1h at room temperature. Odyssey Blocker is expensive but works better than BSA and the like in my hands. 17. Prepare the primary antibody blotting solution as follows: pour 10mL of Odyssey Blocking Solution into a 15mL conical vial. Add 20µL of Tween-20 (0.2% final conc.) and invert the tube twice. Rock the tube for 2min to mix. Add the appropriate dilution of your primary antibody and rock the tube again for 2min. Suggestion: 1:5,000 dilution or follow manufacturer’s directions. 18. Pour off the blocking solution and replace it with the primary antibody blotting solution. Rock the membrane for 1-2h at room temperature in the primary antibody staining solution. Optimal incubation time may vary between antibodies. 19. Pour off the primary antibody solution and replace it with 10mL of 1X TBST (note the addition of Tween-20). Rock membrane for 5min at room temperature. Repeat this step 3X for a total of 4 washes. 20. Prepare the secondary antibody blotting solution in the same manner as in Step 17 using the appropriate dilution of your secondary antibody. If using an antibody with a 680nm fluorophore, add 0.01% SDS to greatly reduce membrane background. Suggestion: 1:5,000 dilution of secondary antibody. 21. Pour off the TBST and replace it with the secondary antibody blotting solution. Gently rock membrane for 1h at room temperature. 22. Pour off the secondary antibody solution and replace it with 1X TBS. Can image membrane wet or dry: dry imaging increases both signal and background. Once dry, the membrane cannot be stripped for re-blotting. Imaging 23. Take the membrane to the LI-COR Odyssey imager. Scan the membrane using the manufacturer’s directions. Note that you must use the appropriate channel for the chosen secondary antibody. 680nm fluorophores are scanned using the 700nm channel. Suggestion: use a scan intensity of 5.0 as a starting point. Band intensity can be increased by increasing exposure post-imaging in the LI-COR software. See manufacturer’s instructions. Reagents Transfer Buffer (1/2 Towbin Buffer) * 3g Tris base (can use Tris-HCl but the amperage of the transfer will need to be monitored due to added salt) * 14.5g Glycine * 200mL Methanol * Dilute to 2L with Milli-Q water 5X TBS (Tris-buffered Saline) Add the following to 300mL of Milli-Q water: * 50mL 1M Tris pH 7.5 * 75mL 5M NaCl * Dilute to 500mL with Milli-Q water and filter sterilize. * Dilute to 1X before use. TBST (TBS with Tween-20) * 50mL of 1X TBS. * 50µL of Tween-20 (0.1% final conc.) * Rock for 2min to mix. Category:Protocols Category:Zebrafish Category:Arabidopsis